The present invention relates to novel dolastatin 10 derivatives having anti-tumor activity and improved side effects, the use of these compounds in the treatment of tumors, pharmaceutical compositions containing those compounds as well as to processes and intermediates for the preparation of these compounds.
Microtubules are known to be the main component of spindles in a mitotic apparatus of eucaryotic cells, and are also involved in many other basic and essential cell functions. Tubulin, a component of microtubules, has attracted our attention for many years as a good molecular target for anticancer therapy (Exp. Opin. Ther. Patents 1999, 9(8): 1069-1081). In fact, tubulin inhibitors such as taxanes and vinca alkaloids are currently used as important anticancer drugs for the treatment of various solid tumors. However, their efficacy is limited and their toxicity such as myelotoxicity is severe because they lack tumor selective activity. Dolastatin 10 is known to be a potent antimitotic peptide, isolated from the marine mollusk Dolabella auricularia, which inhibits tubulin polymerization and is a different chemical class from taxanes and vincas (Curr. Pharm. Des. 1999, 5: 139-162). Preclinical studies of dolastatin 10 have demonstrated activities against a variety of murine and human tumors in cell cultures and animal models. Dolastatin 10 and two synthetic dolastatin derivatives, Cemadotin and TZT-1027 (Drugs of the future 1999, 24(4): 404-409) are currently in Phase I and II clinical trials. While this new class of compounds has yielded certain new anti-tumor agents, these agents have safety drawbacks, such as myelotoxicity, neurotoxicity and some other adverse events.
Surprisingly it has been found that certain dolastatin 10 derivatives having various thio-groups at the dolaproine part show significantly improved anti-tumor activity and therapeutic index in human cancer xenograft models.
Accordingly, the present invention relates to novel compounds of formula I 
wherein
R1, R2 and R3 are each independently selected from hydrogen or (C1-C4)-alkyl;
R4 is selected from the group consisting of
hydrogen;
alkyl optionally substituted with one to three substituents selected from the group consisting of hydroxy, alkoxy, amino, mono- or di-alkylamino, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbonyloxy, carbamoyloxy and halogen;
alkenyl;
alkinyl;
(C3-C7)-cycloalkyl;
aryl optionally substituted with one to three substituents selected from the group consisting of halogen, alkoxycarbonyl, carbamoyl, sulfamoyl,
alkylcarbonyloxy, cyano, mono- or di-alkylamino, alkyl, alkoxy, phenyl, phenoxy, trifluoromethyl, trifluoromethoxy, alkylthio, hydroxy, alkyl-carbonylamino, heterocyclyl, 1,3-dioxolyl, 1,4-dioxolyl, amino and benzyl;
aralkyl wherein the aryl group optionally substituted with one to three substituents selected from the group consisting of halogen, alkoxycarbonyl, carbamoyl, sulfamoyl, alkylcarbonyloxy, cyano, mono- or di-alkylamino, alkyl, alkoxy, phenyl, phenoxy, trifluoromethyl, trifluoromethoxy, alkylthio, hydroxy, alkylcarbonylamino, heterocyclyl, 1,3-dioxolyl, 1,4-dioxolyl, amino and benzyl; and
heterocyclylalkyl;
R5 is selected from the group consisting of
(C1-C6)-alkylamino;
hydroxy;
(C3-C7)-cycloalkylamino optionally substituted by phenyl or benzyl;
arylamino;
aralkylamino having (C1-C4)-alkylene and wherein the aryl group optionally may be substituted with one to three substituents selected from the group consisting of halogen, alkoxycarbonyl, sulfamoyl, alkylcarbonyloxy, carbamoyloxy, cyano, mono- or di-alkylamino, alkyl, alkoxy, phenyl, phenoxy, trifluoromethyl, trifluoromethoxy, alkylthio, hydroxy, alkylcarbonylamino, heterocyclyl, 1,3-dioxolyl, 1,4-dioxolyl, amino and benzyl;
(C1-C4)-alkoxy;
benzhydrazino;
heterocyclyl optionally substituted with one to three substituents selected from the group consisting of benzyl, benzhydryl, alkyl, hydroxy, alkoxy, alkylcarbamoyloxy, amino, mono- or di-alkylamino, acylamino, alkoxy-carbonylamino, phenyl and halogen;
heterocyclylamino;
heterocycloalkylamino wherein the heterocyclyl group optionally may be substituted with one to three substituents selected from the group consisting of benzyl, benzhydryl, alkyl, hydroxy, alkoxy, alkylcarbamoyloxy, amino, dialkylamino, acylamino, alkoxycarbonylamino and halogen;
aralkyloxy and aralkyl, both optionally substituted with one to three substituents from the group consisting of halogen, alkoxycarbonyl, sulfamoyl, alkylcarbonyloxy, cyano, mono- or di-alkylamino, alkyl, alkoxy, phenyl, phenoxy, trifluoromethyl, trifluoromethoxy, alkylthio, hydroxy, alkylcarbonylamino, heterocyclyl, 1,3-dioxolyl, 1,4-dioxolyl, amino, aminosulfonyl and benzyl;
and
n is 0, 1 or 2;
or a pharmaceutical acceptable salt thereof.
These compounds have an anti-tumor activity and are useful for the treatment of malignant diseases, particularly of colorectal cancer, lung cancer, breast cancer, stomach cancer, cervical cancer and bladder cancer.
Unless otherwise indicated the following definitions are set forth to illustrate and define the meaning and scope of the various terms used to described the invention herein.
The term xe2x80x9calkylxe2x80x9d as used herein, alone or in combination, means a straight-chain or branched-chain hydrocarbon group containing a maximum of 12, preferably a maximum of 6, carbon atoms, e.g., methyl, ethyl, n-propyl, 2-methylpropyl (iso-butyl), 1-methylethyl (iso-propyl), n-butyl, and 1,1-dimethylethyl (t-butyl), and more preferably a maximum of 4 carbon atoms. The alkyl group may be unsubstituted or may be substituted with one or more substituents, preferably with one to three substituents, most preferably with one substituent. The substituents are selected from the group consisting of hydroxy, alkoxy, amino, mono- or di-alkylamino, acetoxy, alkylcarbonyloxy, carbamoyloxy, alkoxycarbonyl, carbamoyl or halogen.
The term xe2x80x9calkenylxe2x80x9d as used therein, alone or in combination, refers to a hydrocarbon chain as defined for alkyl having at least one olefinic double bond (including for example, vinyl, allyl and butenyl) and having the general formula CmH2mxe2x88x921 wherein m is an integer greater than 2, preferably m is an integer of 2 to 7.
The term xe2x80x9calkynylxe2x80x9d refers to a hydrocarbon chain as defined for alkyl having at least one triple bond (including for example propynyl, butyn-(1)-yl, etc) and having the general formula CmH2mxe2x88x922 wherein m is an integer greater than 2, preferably m is an integer of 2 to 7.
The term xe2x80x9c(C3-C7)-cycloalkylxe2x80x9d signifies a saturated, cyclic hydrocarbon group with 3-7 carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and the like. The cycloalkyl group may be unsubstituted or substituted with one or more substituents, preferably with one to three substituents, most preferably with one substituent. The substituents are selected from alkyl, phenyl, amino, hydroxy or halogen, preferably is phenyl.
The term xe2x80x9calkylenexe2x80x9d refers to a biradical branched or unbranched hydrocarbon chain containing 1 to 4 carbon atoms, such as methylene (xe2x80x94CH2xe2x80x94), ethylene, propylene, isopropylene and butylene.
The term xe2x80x9carylxe2x80x9d refers to an aromatic carbocyclic radical, i.e. a 6 or 10 membered aromatic or partially aromatic ring, e.g. phenyl (i.e. xe2x80x9cPhxe2x80x9d), naphthyl or tetrahydro-naphthyl, preferably phenyl or naphthyl, and most preferably phenyl. The aryl moiety is optionally substituted with one or more subsituents, preferably with one to three, most preferably one, selected from the group consisting of halogen, preferably fluorine, chlorine, alkoxycarbonyl, (e.g. methoxycarbonyl), alkylcarbonyloxy (e.g., acetoxy), cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, alkylthio, hydroxy, carbamoyloxy, alkylcarbonylamino, heterocyclyl, sulfamoyl (i.e. H2NSO2xe2x80x94), amino, 1,3-dioxolyl, or 1,4-dioxolyl. Especially preferred substituents are alkyl, alkoxy, hydroxy, halogen, amino, alkylamino, dialkylamino, alkylthio, sulfamoyl, benzyl or heterocyclyl.
The term xe2x80x9caralkylxe2x80x9d refers to an aryl group as defined above attached to an alkylene group as defined above. The aryl group of the aralkyl may be substituted with one or more substituents, preferably one to three, more preferably with one to two and most preferably with one substituent selected from the group consisting of halogen, preferably fluorine, chlorine, alkoxycarbonyl, (e.g. methoxycarbonyl), alkylcarbonyloxy (e.g., acetoxy), cyano, alkyl, alkoxy, phenyl, phenoxy, trifluormethyl, trifluormethoxy, alkylthio, hydroxy, carbamoyloxy, alkylcarbonylamino, heterocyclyl, sulfamoyl, amino, 1,3-dioxolyl, or 1,4-dioxolyl. Especially preferred substituents aralkyl, alkoxy, hydroxy, halogen, amino, mono- or di-alkylamino or alkylthio.
The term xe2x80x9cheterocyclylxe2x80x9d refers to a saturated, unsaturated or aromatic monovalent cyclic radical having at one to 3 hetero atoms selected from nitrogen, oxygen or sulfur or a combination thereof, examples of such heterocycles are; furyl, piperidine (preferably piperidin-1-yl, piperidin-4-yl), piperazine (preferably piperazine-1-yl), pyridine, thiophene, thiadiazole, thiazole, benzthiazol, imidazole, tetrahydroisoquinoline and the like. The heterocyclyl may be substituted with one or more substituents, preferably one to three, more preferably with one to two and most preferably with one substituent selected from the group consisting of benzyl, benzhydryl, alkyl, hydroxy, alkoxy, alkyl-carbamoyloxy, amino, dialkylamino, acylamino, alkoxycarbonylamino or halogen.
The term xe2x80x9cheterocyclyl-aminoxe2x80x9d refers to a heterocyclic group as defined above attached via an amino radical, i.e., heterocyclyl-NHxe2x80x94.
The term xe2x80x9cheterocyclyl-alkyl-aminoxe2x80x9d refers to a heterocyclic group as defined above attached via an alkylene group as defined above to the amino radical, i.e. heterocyclyl-alkylene-NHxe2x80x94. The heterocyclylamino may be substituted with one or more substituents, preferably one to three, more preferably with one to two and most preferably with one substituent selected from the group consisting of benzyl, benzhydryl, alkyl, hydroxy, alkoxy, alkylcarbamoyloxy, amino, mono- or di-alkylamino, acylamino, alkoxycarbonyl-amino or halogen. Especially preferred substituents are alkyl, hydroxy, alkylcarbamoyloxy, amino, dialkylamino, acylamino, alkylcarbonylamino or halogen.
The term xe2x80x9caminoxe2x80x9d refers to the group xe2x80x94NH2 and includes amino groups which are further substituted by a lower alkyl group(s), or protected by a group known in the art such as a benzoxycarbonyl group, acetyl group, alkoxycarbonyl group or benzyl group and the like.
The term xe2x80x9ccycloalkylaminoxe2x80x9d refers to cycloalkyl group as defined above attached to a structure via an amino radical, such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and the like. The cycloalkylamino group may be unsubstituted or substituted with one or more substituents, preferably one to three, more preferably with one to two and most preferably with one substituent. The substituents are preferably phenyl or benzyl.
The term xe2x80x9carylaminoxe2x80x9d refers to an aryl group as defined above attached to a parent structure via an amino radical, i.e., aryl-NHxe2x80x94.
The term xe2x80x9caralkylaminoxe2x80x9d refers to an aryl group as defined above attached to a parent structure via an alkylene-amino radical, i.e., aralkyl-NHxe2x80x94. The aralkylamino group may be optionally substituted with a lower alkyl group, preferably a methyl group, i.e., aralkyl-NCH3xe2x80x94.
The term xe2x80x9cacetoxyxe2x80x9d refers to the group xe2x80x94Oxe2x80x94OCxe2x80x94CH3.
The term xe2x80x9ccarbamoylxe2x80x9d refers to the group xe2x80x94COxe2x80x94NH2 and the term xe2x80x9ccarbamoyloxyxe2x80x9d to the group xe2x80x94Oxe2x80x94COxe2x80x94NH.
The term xe2x80x9calkylcarbamoyloxyxe2x80x9d refers to an alkyl group as defined above attached to a parent structure via a carbymoyloxy radical, i.e., xe2x80x94Oxe2x80x94COxe2x80x94NH-alkyl.
The term xe2x80x9calkylcarbonyloxyxe2x80x9d refers to an alkyl group as defined above attached to a parent structure via a carbonyloxy radical, i.e., xe2x80x94Oxe2x80x94CO-alkyl.
The term xe2x80x9calkoxyxe2x80x9d refers to the group Rxe2x80x2xe2x80x94Oxe2x80x94, wherein Rxe2x80x2 is an alkyl group as defined above.
The term xe2x80x9caralkyloxyxe2x80x9d refers to the group Yxe2x80x94Oxe2x80x94, wherein Y is aralkyl group as defined above.
The term xe2x80x9calkylthioxe2x80x9d refers to the group Rxe2x80x94Sxe2x80x94, wherein R is an alkyl group as defined above.
The term xe2x80x9chalogenxe2x80x9d refers to fluorine, bromine, iodine and chlorine.
In the present invention, the expression xe2x80x9coptionally substituted withxe2x80x9d means that substitution can occur at one or more positions, preferably at one to three positions, and, unless otherwise indicated, that the substituents are independently selected from the specified options.
xe2x80x9cPharmaceutically acceptable saltxe2x80x9d refers to conventional acid-addition salts or base-addition salts which retain the biological effectiveness and properties of the compounds of formula I and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Sample base-addition salts include those derived from potassium, sodium, ammonium, and quarternary ammonium hydroxide, such as for example tetramethylammonium hydroxide.
xe2x80x9cPharmaceutically acceptable,xe2x80x9d such as pharmaceutically acceptable carrier, excipient, prodrug, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered.
xe2x80x9cPharmaceutically active metabolitexe2x80x9d means a metabolic product of a compound of formula I which is pharmaceutically acceptable and effective.
In one embodiment, the present invention is directed to a compound of formula I 
wherein
R1, R2 and R3 are each independently selected from hydrogen or (C1-C4)-alkyl;
R4 is selected from the group consisting of
hydrogen;
alkyl optionally substituted with one to three substituents selected from the group consisting of hydroxy, alkoxy, amino, mono- or di-alkylamino, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbonyloxy and halogen;
alkenyl;
alkinyl;
(C3-C7)-cycloalkyl;
aryl optionally substituted with one to three substituents selected from the group consisting of halogen, alkoxycarbonyl, sulfamoyl, alkylcarbonyloxy, cyano, mono- or di-alkylamino, alkyl, alkoxy, phenyl, phenoxy, trifluoromethyl, trifluoromethoxy, alkylthio, hydroxy, alkylcarbonylamino, heterocyclyl, 1,3-dioxolyl, 1,4-dioxolyl, amino and benzyl;
aralkyl wherein the aryl group optionally substituted with one to three substituents selected from the group consisting of halogen, alkoxycarbonyl, sulfamoyl, alkylcarbonyloxy, cyano, mono- or di-alkylamino, alkyl, alkoxy, phenyl, phenoxy, trifluoromethyl, trifluoromethoxy, alkylthio, hydroxy, alkylcarbonylamino, heterocyclyl, 1,3-dioxolyl, 1,4-dioxolyl, amino and benzyl; and
heterocyclylalkyl;
R5 is selected from the group consisting of
(C1-C6)-alkylamino;
hydroxy;
(C3-C7)-cycloalkylamino optionally substituted by phenyl or benzyl;
arylamino;
aralkylamino having (C1-C4)-alkylene and wherein the aryl group optionally may be substituted with one to three substituents selected from the group consisting of halogen, alkoxycarbonyl, sulfamoyl, alkylcarbonyloxy, cyano, mono- or di-alkylamino, alkyl, alkoxy, phenyl, phenoxy, trifluoromethyl, trifluoromethoxy, alkylthio, hydroxy, alkylcarbonylamino, heterocyclyl, 1,3-dioxolyl, 1,4-dioxolyl, amino and benzyl;
(C1-C4)-alkoxy;
benzhydrazino;
heterocyclyl optionally substituted with one to three substituents selected from the group consisting of benzyl, benzhydryl, alkyl, hydroxy, alkoxy, alkylcarbamoyloxy, amino, mono- or di-alkylamino, acylamino, alkoxy-carbonylamino, phenyl and halogen;
heterocyclylamino;
heterocycloalkylamino wherein the heterocyclyl group optionally may be substituted with one to three substituents selected from the group consisting of benzyl, benzhydryl, alkyl, hydroxy, alkoxy, alkylcarbamoyloxy, amino, dialkylamino, acylamino, alkoxycarbonylamino and halogen;
aralkyloxy and aralkyl, both optionally substituted with one to three substituents from the group consisting of halogen, alkoxycarbonyl, sulfamoyl, alkylcarbonyloxy, cyano, mono- or di-alkylamino, alkyl, alkoxy, phenyl, phenoxy, trifluoromethyl, trifluoromethoxy, alkylthio, hydroxy, alkylcarbonylamino, heterocyclyl, 1,3-dioxolyl, 1,4-dioxolyl, amino, aminosulfonyl and benzyl;
and
n is 0, 1 or 2;
or a pharmaceutical acceptable salt thereof.
Preferably, the present invention relates to compounds of the above formula (I), wherein R4 is hydrogen; alkyl optionally substituted with one to three substituents selected from the group consisting of hydroxy, amino, mono- or di-alkylamino, carbamoyl, acetoxy, carbamoyloxy or carboxy; alkenyl; alkinyl; (C3-C7)-cycloalkyl; aryl optionally substituted with one to three substituents selected from the group consisting of alkyl, alkoxy, hydroxy, halogen, amino, mono- or di-alkylamino, alkylthio or alkylcarbonylamino; aralkyl with the aryl group optionally substituted with one to three substituents selected from the group consisting of alkyl, alkoxy, hydroxy, halogen, amino, mono- or di-alkylamino, or alkylthio; or heterocyclylalkyl.
More preferably, the present invention relates to compounds of the above formula (I), wherein R4 is phenyl, methyl, t-butyl, 4-tButylphenyl, 4-methoxyphenyl, 2-aminoethyl, 2-dimethylaminoethyl, ZHNCH2CH2xe2x80x94 (xe2x80x9cZxe2x80x9d is the group benzyloxycarbonyl), 4-methylthiophenyl, cyclohexyl, 2-, 3-, or 4-hydroxyphenyl, 4-acetoaminophenyl, 4-fluorophenyl, ethyl, i-propyl, benzyl, 2-acetoxyethyl, ethylcarbamoyloxyethyl, diethylcarbamoylmethyl, phenylethyl, allyl, n-pentyl, 2-naphtyl, 4-fluorobenzyl, 2-furylmethyl or 2-hydroxyethyl.
Most preferably, the present invention relates to compounds of the above formula (I), wherein R4 is phenyl; 4-hydroxyphenyl (R); 4-acetoaminophenyl; tertia-butyl; (R); ethyl; isopropyl; t-butyl; benzyl; 3-hydroxyphenyl 2-hydroxyphenyl; 2-acetoxyethyl; allyl; n-pentyl, 2-hydroxyethyl or methyl.
In another preferred embodiment of a compound of formula (I), R5 is (C1-C6)-alkylamino; hydroxy; (C3-C7)-cycloalkylamino optionally substituted by phenyl or benzyl; arylamino; aralkylamino having (C1-C4)-alkylene and the aryl group optionally substituted with one to three substituents selected from the group consisting of H2NSO2xe2x80x94, hydroxy, alkyl, benzyl, alkoxy, carbamoyloxy or heterocyclyl; (C1-C4)-alkoxy; benzhydrazino; heterocyclyl optionally substituted by benzyl or benzhydryl; heterocyclylamino; heterocycloalkyamino with the heterocyclyl group optionally substituted with one to three substituents selected from the group consisting of alkyl, hydroxy, alkoxy, alkylcarbamoyloxy, amino, dialkylamino, acylamino, alkoxycarbonylamino or halogen; or aralkyloxy and aralkyl both optionally substituted with one to three substituents from the group consisting of halogen, alkoxycarbonyl, sulfamoyl, alkylcarbonyloxy, cyano, mono- or di-alkylamino, alkyl, alkoxy, phenyl, phenoxy, trifluoromethyl, trifluoromethoxy, alkylthio, hydroxy, alkylcarbonylamino, heterocyclyl, 1,3-dioxolyl, 1,4-dioxolyl, amino, aminosulfonyl or benzyl.
More preferably, the present invention relates to compounds of the above formula (I), wherein R5 is phenylethylamino; phenylethoxy; benzyloxy; 2-naphtylmethylamino; benzyl-piperazino; 1,2,3,4-tetrahydroisoquinolino; t-butoxy; hydroxy; 4-H2NSO2PhCH2CH2; 2-, 3- or 4-hydroxyphenylethylamino; 2-, 3- or 4-hydroxyphenylethyl-N-methylamino; N-benzylphenethylamino; 4-t-butylbenzylamino; benzylamino; N-methylphenethylamino; 4-benzhydrylpiperazino; 2-phenylcyclopropylamino; thienylethylamino; 2-pyridylethylamino; 5-ethylpyrazol; 4,3-dimethoxyphenylethylamino; benzylhydrazino; benzothiazol-2-ylmethyl-amino; 2-pyridin-4-yl-amino; 3,4-dimethoxy-phenyl-ethyl-methyl-amino;, bezothiazol-2-ylmethyl-amino; 2-pyridin-3-yl-ethylamino; pyridin-4-ylmethyl-amino; thiazol-2-ylamino; naphtalen-2-ylamino; 4-chloro-phenyl-ethylamino; 4-methoxy-phenyl-ethylamino; 4-(1,2,3)thiadiazol-4-yl-benzylamino; 2-cyclohexylamino or 1-benzyl-piperidin-4-ylamino.
Most preferably, the present invention relates to compounds of the above formula (I) wherein R5 is phenylethylamino, 4,3-dimethoxyphenylethylamino, thienylethylamino, 2-pyridylethylamino, 4-hydroxyphenylethylamino, N-methylphenethylamino, 2-hydroxy-phenylethylamino, 3-hydroxyphenylethylamino, 2-hydroxyphenylethyl-N-methylamino, 3-hydroxyphenylethyl-N-methylamino, 4-hydroxyphenylethyl-N-methylamino or benzylhydrazino.
In another preferred embodiment R1 and R2 are each independently selected from (C1-C4)-alkyl. Most preferably, R1 and R2 are methyl.
Additionally preferred are compounds of formula (I) wherein R1 and R2 are methyl, R3 is hydrogen and n is 0. Examples of such compounds are:
a) N-[1-({1-sec-Butyl-2-methoxy-4-oxo-4-[2-(2-phenethylcarbamoyl-1-phenylsulfanyl-ethyl)-pyrrolidin-1-yl]-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
b) N-[1-({1-sec-Butyl-2-methoxy-4-[2-(1-methylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
c) N-[1-({1-sec-Butyl-4-[2-(1-(S)-tert-butylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
d) N-{1-[(1-sec-Butyl-4-{2-[1-(4-tert-butyl-phenylsulfanyl)-2-phenethylcarbabamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
e) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-(4-methoxy-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
f) 3-[1-(4-{[2-(2-Dimethylamino-3-methyl-butyrylamino)-3-methyl-butyryl]-methyl-amino}-3-methoxy-5-methyl-heptanoyl)-pyrrolidin-2-yl]-3-methylsulfanyl-propionic acid phenethyl ester,
g) 3-[1-(4-{[2-(2-Dimethylamino-3-methyl-butyrylamino)-3-methyl-butyryl]-methyl-amino}-3-methoxy-5-methyl-heptanoyl)-pyrrolidin-2-yl]-3-methylsulfanyl-propionic acid benzyl ester,
h) N-(1-{[1-sec-Butyl-2-methoxy-4-(2-{1-methylsulfanyl-2-[(naphthalen-2-ylmethyl)-carbamoyl]-ethyl}-pyrrolidin-1-yl)-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
i) N-{1-[(4-{2-[1-(2-Amino-ethylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-1-sec-butyl-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
j) N-{1-[(4-{2-[3-(4-Benzyl-piperazin-1-yl)-1-methylsulfanyl-3-oxo-propyl]-pyrrolidin-1-yl}-1-sec-butyl-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
k) N-{1-[(1-sec-Butyl-4-{2-[3-(3,4-dihydro-1H-isoquinolin-2-yl)-1-methylsulfanyl-3-oxo-propyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
l) N-{1-[(1-sec-Butyl-4-{2-[1-(2-dimethylamino-ethylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
m) (2-{1-[1-(4-{[2-(2-Dimethylamino-3-methyl-butyrylamino)-3-methyl-butyryl]-methyl-amino}-3-methoxy-5-methyl-heptanoyl)-pyrrolidin-2-yl]-2-phenethylcarbamoyl-ethylsulfanyl}-ethyl)-carbamic acid benzyl ester,
n) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-(4-methylsulfanyl-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
o) N-[1-({1-sec-Butyl-4-[2-(1-cyclohexylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
p) N-{1-[(1-sec-Butyl-4-{2-[1-(S)-(4-hydroxy-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
q) N-{1-[(1-sec-Butyl-4-{2-[1-(R)-(4-hydroxy-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
r) N-{1-[(4-{2-[1-(4-Acetylamino-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-1-sec-butyl-2-methoxy-4-oxo-butyl)-methyl -carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
s) N-{1-[(1-sec-Butyl-4-{2-[1-(4-fluoro-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
t) N-[1-({1-sec-Butyl-4-[2-(1-(R)-tert-butylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
u) N-[1-({1-sec-Butyl-4-[2-(1-ethylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
v) N-[1-({1-sec-Butyl-4-[2-(1-isopropylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
w) N-[1-({1-sec-Butyl-4-[2-(1-tert-butylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
x) N-[1-({4-[2-(1-Benzylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-1-sec-butyl-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
y) N-{1-[(1-sec-Butyl-4-{2-[1-(2-hydroxy-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
z) N-{1-[(1-sec-Butyl-4-{2-[1-(3-hydroxy-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
aa) N-{1-[(1-sec-Butyl-4-{2-[1-(2-hydroxy-ethylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
bb) Acetic acid 2-{1-[1-(4-{[2-(2-dimethylamino-3-methyl-butyrylamino)-3-methyl-butyryl]-methyl-amino}-3-methoxy-5-methyl-heptanoyl)-pyrrolidin-2-yl]-2-phenethylcarbamoyl-ethylsulfanyl}-ethyl ester,
cc) 3-[1-(4-{[2-(2-Dimethylamino-3-methyl-butyrylamino)-3-methyl-butyryl]-methyl-amino}-3-methoxy-5-methyl-heptanoyl)-pyrrolidin-2-yl]-3-methylsulfanyl-propionic acid tert-butyl ester,
dd) 3-[1-(4-{[2-(2-Dimethylamino-3-methyl-butyrylamino)-3-methyl-butyryl]-methyl-amino}-3-methoxy-5-methyl-heptanoyl)-pyrrolidin-2-yl]-3-methylsulfanyl-propionic acid,
ee) N-(1-{[1-sec-Butyl-2-methoxy-4-(2-{1-methylsulfanyl-2-[2-(4-sulfamoyl-phenyl)-ethylcarbamoyl]-ethyl}-pyrrolidin-1-yl)-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
ff) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(4-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
gg) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[2-(methyl-phenethyl-carbamoyl)-1-methylsulfanyl-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
hh) N-{1-[(4-{2-[3-(4-Benzhydryl-piperazin-1-yl)-1-methylsulfanyl-3-oxo-propyl]-pyrrolidin-1-yl}-1-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
ii) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(2-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
jj) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
kk) N-{1-[(4-{2-[2-(Benzyl-phenethyl-carbamoyl)-1-methylsulfanyl-ethyl]-pyrrolidin-1-yl}-1-sec-butyl-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
ll) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(2-phenyl-cyclopropylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
mm) N-{1-[(1-sec-Butyl-4-{2-[2-(4-tert-butyl-benzylcarbamoyl)-1-methylsulfanyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
nn) N-[1-({4-[2-(2-Benzylcarbamoyl-1-methylsulfanyl-ethyl)-pyrrolidin-1-yl]-1-sec-butyl-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
oo) N-{1-[(4-{2-[2-(Nxe2x80x2-Benzyl-hydrazinocarbonyl)-1-methylsulfanyl-ethyl]-pyrrolidin-1-yl}-1-sec-butyl-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
pp) N-[1-({1-sec-Butyl-2-methoxy-4-oxo-4-[2-(2-phenethylcarbamoyl-1-phenethylsulfanyl-ethyl)-pyrrolidin-1-yl]-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
qq) N-[1-({4-[2-(1-Allylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-1-sec-butyl-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
rr) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(2-pyridin-4-yl-ethylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
ss) N-(1-{[4-(2-{2-[(Benzothiazol-2-ylmethyl)-carbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-1-sec-butyl-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
tt) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(2-thiophen-2-yl-ethylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
uu) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(2-pyridin-3-yl-ethylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
vv) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(2-pyridin-2-yl-ethylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
ww) N-(1-{[1-sec-Butyl-2-methoxy-4-(2-{1-methylsulfanyl-2-[(pyridin-4-ylmethyl)-carbamoyl]-ethyl}-pyrrolidin-1-yl)-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
xx) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3H-imidazol-4-yl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
yy) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(thiazol-2-ylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
zz) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(naphthalen-2-ylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
aaa) N-[1-({1-sec-Butyl-4-[2-(2-cyclohexylcarbamoyl-1-methylsulfanyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
bbb) N-[1-({1-sec-Butyl-4-[2-(2-{[2-(3,4-dimethoxy-phenyl)-ethyl]-methyl-carbamoyl}-1-methylsulfanyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
ccc) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3,4-dimethoxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
ddd) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(4-chloro-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
eee) N-[1-({1-sec-Butyl-2-methoxy-4-oxo-4-[2-(1-pentylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
fff) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-(naphthalen-2-ylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
ggg) N-{1-[(1-sec-Butyl-4-{2-[1-(4-fluoro-benzylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
hhh) N-{1-[(1-sec-Butyl-4-{2-[1-(furan-2-ylmethylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
iii) N-(1-{[1-sec-Butyl-2-methoxy-4-(2-{2-[2-(4-methoxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
jjj) N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(4-[1,2,3]thiadiazol-4-yl-benzylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
kkk) N-{1-[(4-{2-[2-(1-Benzyl-piperidin-4-ylcarbamoyl)-1-methylsulfanyl-ethyl]-pyrrolidin-1-yl}-1-sec-butyl-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
lll) N-(1-{[1-sec-Butyl-4-(2-{1-tert-butylsulfanyl-2-[2-(4-hydroxy-phenyl)-ethylcarbamoyl]-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
mmm) N-(1-{[1-sec-Butyl-4-(2-{1-tert-butylsulfanyl-2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
nnn) N-(1-{[1-sec-Butyl-4-(2-{1-tert-butylsulfanyl-2-[2-(2-hydroxy-phenyl)-ethylcarbamoyl]-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
ooo) N-(1-{[1-sec-Butyl-4-(2-{1-dimethylcarbamoylmethylsulfanyl-2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
ppp) N-[1-({1-sec-Butyl-4-[2-(1-dimethylcarbamoylmethylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide, and
qqq) Ethyl-carbamic acid 2-{1-[1-(4-{[2-(2-dimethylamino-3-methyl-butyrylamino)-3-methyl-butyryl]-methyl-amino}-3-methoxy-5-methyl-heptanoyl)-pyrrolidin-2-yl]-2-phenethylcarbamoyl-ethylsulfanyl}-ethyl ester.
When R1 and R2 are methyl, R3 is hydrogen and n is an integer of 0, preferred compounds of formula (I) are those for which R4 is phenyl, 4-hydroxyphenyl (R), 4-AcNHPh- (i.e., 4-acetoaminophenyl), t-butyl (R), ethyl, i-propyl, , t-butyl, benzyl, 3-hydroxyphenyl, 2-hydroxyphenyl, 2-hydroxyethyl, 2-acetoxyethyl, allyl or n-pentyl and R5 is phenylethylamino. In particular, the following compounds of formula (I) are preferred in the present invention;
N-[1-({1-sec-Butyl-2-methoxy-4-oxo-4-[2-(2-phenethylcarbamoyl-1-phenylsulfanyl-ethyl)-pyrrolidin-1-yl]-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-{1-[(1-sec-Butyl-4-{2-[1-(R)-(4-hydroxy-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
N-{1-[(4-{2-[1-(4-Acetylamino-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-1-sec-butyl-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
N-[1-({1-sec-Butyl-4-[2-(1-(R)-tert-butylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-[1-({1-sec-Butyl-4-[2-(1-ethylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-[1-({1-sec-Butyl-4-[2-(1-isopropylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-[1-({1-sec-Butyl-4-[2-(1-tert-butylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-[1-({4-[2-(1-Benzylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-1-sec-butyl-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-{1-[(1-sec-Butyl-4-{2-[1-(3-hydroxy-phenylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
N-{1-[(1-sec-Butyl-4-{2-[1-(2-hydroxy-ethylsulfanyl)-2-phenethylcarbamoyl-ethyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
Acetic acid 2-{1-[1-(4-{[2-(2-dimethylamino-3-methyl-butyrylamino)-3-methyl-butyryl]-methyl-amino}-3-methoxy-5-methyl-heptanoyl)-pyrrolidin-2-yl]-2-phenethylcarbamoyl-ethylsulfanyl}-ethyl ester,
N-[1-({4-[2-(1-Allylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-1-sec-butyl-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide, and
N-[1-({1-sec-Butyl-2-methoxy-4-oxo-4-[2-(1-pentylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide.
Other preferred compounds of formula (I) are those wherein R1 and R2 are methyl, R3 is hydrogen and n is 0.
Other preferred compounds of formula (I) are those for which R4 is methyl and R5 is selected from 4-hydroxyphenylethylamino; N-methylphenethylamino; 2-hydroxyphenylethylamino; 3-hydroxyphenylethylamino; benzylhydrazino; 4,3-dimethoxyphenylethylamino; thienylethylamino; and 2-pyridylethylamino. Examples of such compounds include:
N-(1-{[1-sec-Butyl-4-(2-{2-[2-(4-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[2-(methyl-phenethyl-carbamoyl)-1-methylsulfanyl-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
N-(1-{[1-sec-Butyl-4-(2-{2-[2-(2-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
N-{1-[(4-{2-[2-(Nxe2x80x2-Benzyl-hydrazinocarbonyl)-1-methylsulfanyl-ethyl]-pyrrolidin-1-yl}-1-sec-butyl-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(2-thiophen-2-yl-ethylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(2-pyridin-3-yl-ethylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
N-{1-[(1-sec-Butyl-2-methoxy-4-{2-[1-methylsulfanyl-2-(2-pyridin-2-yl-ethylcarbamoyl)-ethyl]-pyrrolidin-1-yl}-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide, and
N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3,4-dimethoxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide.
Other compounds of interest include compounds of formula (I) wherein R1 and R2 are methyl, R3 is hydrogen and n is 1. An example of such a compound is N-[1-({1-sec-Butyl-4-[2-(1-methanesulfinyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide.
Other compounds of interest include compounds of formula (1) wherein R1 and R2 are methyl, R3 is hydrogen and n is 2. An example of such a compound is the compound N-[1-({1-sec-Butyl-4-[2-(1-methanesulfonyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide.
Another preferred embodiment of the present invention is a compound of formula (I) wherein R1 is methyl, R2 and R3 are hydrogen and n is 0. Examples of such compounds are selected from the group consisting of.
a) N-[1-({1-sec-Butyl-4-[2-(1-ethylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
b) N-[1-({1-sec-Butyl-2-methoxy-4-oxo-4-[2-(2-phenethylcarbamoyl-1-phenylsulfanyl-ethyl)-pyrrolidin-1-yl]-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
c) N-[1-({1-sec-Butyl-4-[2-(1-tert-butylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
d) N-[1-({1-sec-Butyl-2-methoxy-4-[2-(1-methylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
e) N-[1-({1-sec-Butyl-4-[2-(1-isopropylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
f) N-{1-[(1-sec-Butyl-2-methoxy-4-oxo-4-{2-[2-phenethylcarbamoyl-1-(2-methyl-propane-2-sulfonyl)-ethyl]-pyrrolidin-1-yl}-butyl)-methyl-carbamoyl]-2-methyl-propyl}-3-methyl-2-methylamino-butyramide,
g) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-3-methyl-2-methylamino-butyramide, and
h) N-(1-{[1-sec-Butyl-4-(2-{1-tert-butylsulfanyl-2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-3-methyl-2-methylamino-butyramide.
When R1 is methyl, R2 and R3 are hydrogen and n is 0, preferred compounds of formula (I) are those for which R4 is ethyl, phenyl, t-butyl, methyl, i-propyl and R5 is phenylethylamino or 3-hydroxyphenylethylamino. In particular, the following compounds of formula (I) are preferred:
N-[1-({1-sec-Butyl-4-[2-(1-ethylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
N-[1-({1-sec-Butyl-2-methoxy-4-oxo-4-[2-(2-phenethylcarbamoyl-1-phenylsulfanyl-ethyl)-pyrrolidin-1-yl]-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
N-[1-({1-sec-Butyl-2-methoxy-4-[2-(1-methylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
N-[1-({1-sec-Butyl-4-[2-(1-isopropylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide, and
N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-ethyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-3-methyl-2-methylamino-butyramide.
Another preferred embodiment of the present invention concerns compound of formula (I) wherein R1 is methyl, R2 and R3 are hydrogen and n is 2. Examples of such compounds include:
a) N-[1-({1-sec-Butyl-4-[2-(1-ethanesulfonyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
b) N-[1-({4-[2-(1-Benzenesulfonyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-1-sec-butyl-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
c) N-[1-({1-sec-Butyl-4-[2-(1-methanesulfonyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide, and
d) N-{1-[(1-sec-Butyl-2-methoxy-4-oxo-4-{2-[2-phenethylcarbamoyl-1-(propane-2-sulfonyl)-ethyl]-pyrrolidin-1-yl}-butyl)-methyl-carbamoyl]-2-methyl-propyl}-3-methyl-2-methylamino-butyramide.
When R1 is methyl, R2 and R3 are hydrogen, and n is 2, preferably R4 is methyl and R5 is phenylethylamino. An example of such a compound is
N-[1-({1-sec-Butyl-4-[2-(1-methanesulfonyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide.
Another preferred embodiment of the present invention concerns compounds of formula (I) wherein R1 and R3 are methyl, R2 is hydrogen and n is 0. Examples of such compounds are selected from the group consisting of:
a) N-[1-({1-sec-Butyl-2-methoxy-4-[2-(1-methylsulfanyl-2-phenethylcarbamoyl-propyl)-pyrrolidin-1-yl]-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-methylamino-3-methyl-butyramide, and
b) N-[-({1-sec-Butyl-4-[2-(1-tert-butylsulfanyl-2-phenethylcarbamoyl-propyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-methylamino-3-methyl-butyramide.
Another preferred embodiment of the present invention concerns compound of formula (I) wherein R1, R2 and R3 are methyl and n is 0. Examples of such compounds are selected from the group consisting of:
a) N-[1-({1-sec-Butyl-2-methoxy-4-[2-(1-methylsulfanyl-2-phenethylcarbamoyl-propyl)-pyrrolidin-1-yl]-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
b) N-[1-({1-sec-Butyl-4-[2-(1-tert-butylsulfanyl-2-phenethylcarbamoyl-propyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
c) N-{1-[(1-sec-Butyl-4-{2-[1-(2-hydroxy-ethylsulfanyl)-2-phenethylcarbamoyl-propyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
d) N-{1-[(1-sec-Butyl-4-{2-[1-(4-hydroxy-phenylsulfanyl)-2-phenethylcarbamoyl-propyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
e) N-{1-[(1-sec-Butyl-4-{2-[1-(3-hydroxy-phenylsulfanyl)-2-phenethylcarbamoyl-propyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
f) N-{1-[(1-sec-Butyl-4-{2-[1-(2-hydroxy-phenylsulfanyl)-2-phenethylcarbamoyl-propyl]-pyrrolidin-1-yl}-2-methoxy-4-oxo-butyl)-methyl-carbamoyl]-2-methyl-propyl}-2-dimethylamino-3-methyl-butyramide,
g) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(4-hydroxy-phenyl)-ethylcarbamoyl]-1-t-butylsulfanyl-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
h) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-1-t-butylsulfanyl-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
i) N-(1-{[-sec-Butyl-4-(2-{2-[2-(2-hydroxy-phenyl)-ethylcarbamoyl]-1-t-butylsulfanyl-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
j) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(4-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
k) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
l) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(2-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
m) N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-1-pentylsulfanyl-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
n) N-[1-({1-sec-Butyl-4-[2-(2-{[2-(3-hydroxy-phenyl)-ethyl]-methyl-carbamoyl}-1-methylsulfanyl-propyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
o) N-[1-({1-sec-Butyl-4-[2-(1-ethylsulfanyl-2-{[2-(3-hydroxy-phenyl)-ethyl]-methyl-carbamoyl}-propyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
p) N-(1-{[1-sec-Butyl-4-(2-{1-ethylsulfanyl-2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide, and
q) Ethyl-carbamic acid 3-(2-{3-[1-(4-{[2-(2-dimethylamino-3-methyl-butyrylamino)-3-methyl-butyryl]-methyl-amino}-3-methoxy-5-methyl-heptanoyl)-pyrrolidin-2-yl]-2-methyl-3-methylsulfanyl-propionylamino}-ethyl)-phenyl ester.
Preferably, when R1, R2 and R3 are methyl or ethyl (preferably methyl) and n is 0, R4 is methyl or ethyl and R5 is phenylethylamino, 3-hydroxyphenylethylamino or 3-hydroxyphenylethyl-N-methyamino. An example of such a compound includes
N-[1-({1-sec-Butyl-2-methoxy-4-[2-(1-methylsulfanyl-2-phenethylcarbamoyl-propyl)-pyrrolidin-1-yl]-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
N-[1-({1-sec-Butyl-4-[2-(2-{[2-(3-hydroxy-phenyl)-ethyl]-methyl-carbamoyl}-1-methylsulfanyl-propyl)-pyrrolidin-1-yl]-2-.methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-[1-({1-sec-Butyl-4-[2-(1-ethylsulfanyl-2-{[2-(3-hydroxy-phenyl)-ethyl]carbamoyl}-propyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide, and
N-(1-{[1-sec-Butyl-4-(2-{1-ethylsulfanyl-2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide.
All of the stereoisomers in the formula (I) are included in the scope of the invention. However, compounds having the stereostructural formula (I-1), 
wherein R1, R2, R3,R4, R5 and n are as defined above, as well as pharmaceutically acceptable salts thereof, are preferred.
Compounds of formula (I) and formula (I-1) wherein the R4S(O)n group has an R-configuration and the R3 group has an S-configuration have particularly advantageous antitumor activity.
The compounds of the invention are effective at inhibiting or preventing the growth of tumors in premalignant and malignant cells and are useful in preventing carcinomas from forming solid tumors. The compounds are particularly useful in the treatment of colorectal cancer, lung cancer, breast cancer, stomach cancer, cervical cancer and bladder cancer. The compounds of this invention can be used to treat such tumors, to retard the development of such tumors, and to prevent the increase in number of tumors.
The anticancer therapeutic activity of compounds of this invention is demonstrated by various standard in vitro assays. The assays described below and in the examples are known to indicate antitumor and anticancer activity and are assays used by those skilled in the art to assess compounds for cancer therapeutics.
Compounds of this invention have the structure depicted in formula I, and anticancer activity as determined by any standard assay, especially assays for apoptosis. The compounds are particularly effective to induce apoptosis in carcinoma cells, causing the death of the cell. Thus a compound has the desired activity if the compound causes carcinoma cells to die when the cells are exposed to the compounds. Carcinoma cells for assays (for example breast, lung, colorectal, etc.) are readily obtained from cell depositories such as the American Type Culture Collection (ATCC) or may be isolated by skilled persons from cancer patients. The type of cancer against which the compound is most active is determined by the type of cell used in the assays.
Carcinoma cells, grown in culture, may be incubated with a specific test compound and changes in cell viability may be determined for example, by dyes which selectively stain dead cells or by optical density (O.D.) measurement. If more than 10% of cells have died, the compound is active in inducing apoptosis. The compounds may not directly kill the cells (cellular toxicity) but may modulate certain intra- or extracellular events which result in apoptosis.
The anticancer activity of the compounds of this invention may also be determined by assays that access the effects of compounds on cell growth and differentiation. Cell growth inhibition may be determined by adding the test compound to carcinoma cells in culture with dyes or radioactive precursors, and determining by microscopic cell counting, scintillation counting, or O.D. measurement whether the number of cells has increased over the incubation period. If the number of cells has not increased, growth has been inhibited and the compound is regarded as having therapeutic activity. Similarly, the proportion of cells which have become differentiated after addition of a test compound may be determined by known methods (ie. measuring oxidative burst in HL-60 cells, an indicator of differentiation, by NBT). If 10% or more cells have differentiated, then the compound is regarded as having therapeutic activity.
In vivo assays are also useful to demonstrate anticancer activity. Compounds of this invention are effective in reducing the size and/or the number of tumors in laboratory animals such as mice in which tumor growth has been induced. The type of tumor indicates the type of cancer against which primary activity is expected. Specific tumors may be induced by perturbing specific tissues with carcinogens, or by injecting specific types of carcinoma cells. The compounds of the present invention show significant prophylactic and therapeutic activity when evaluated against NMU-induced mammary (breast) tumors in rats. Surprisingly the doses and regimens which are effective are free of significant toxicity. The compounds also show efficacy in reducing number of tumors during the course of the experiment (i.e. chemoprevention) at doses and regimens not associated with toxicity. Furthermore, the compounds are therapeutically active, i.e. are able to effect regression of established first primary tumors. The compounds are also preventitive, i.e. able to significantly prevent formation of new tumors.
In Vitro Assay
Antiproliferative activity assay was carried out as follows. A single suspension of tumor cells was inoculated to the serially diluted 96-well microtestplate. Then the testplate was incubated in the 5% CO2 ambience at 37xc2x0 C. for 4 days (2-3xc3x97103 cells/well). The degree of cell growth in a monolayer was measured by using WST-8 (Dojindo, Japan). IC50 values of drugs against tumor cells were calculated as the concentration of drug yielding 50% OD of the control growth. The results are shown in the following table I.
The maximum tolerated doses (MTD) of the following representative compounds
N-[1-({1-sec-Butyl-2-methoxy-4-[2-(1-methylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
N-[1-({1-sec-Butyl-4-[2-(1-methanesulfonyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
N-[1-({1-sec-Butyl-4-[2-(1-isopropylsulfanyl-2-phenethylcarbamoyl-ethyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-3-methyl-2-methylamino-butyramide,
N-[1-({1-sec-Butyl-4-[2-(2-{[2-(3-hydroxy-phenyl)-ethyl]-methyl-carbamoyl}-1-methylsulfanyl-propyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-[1-({1-sec-Butyl-4-[2-(1-ethylsulfanyl-2-{[2-(3-hydroxy-phenyl)-ethyl]-methyl-carbamoyl}-propyl)-pyrrolidin-1-yl]-2-methoxy-4-oxo-butyl}-methyl-carbamoyl)-2-methyl-propyl]-2-dimethylamino-3-methyl-butyramide,
N-(1-{[1-sec-Butyl-4-(2-{2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-1-methylsulfanyl-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide,
N-(1-{[1-sec-Butyl-4-(2-{1-ethylsulfanyl-2-[2-(3-hydroxy-phenyl)-ethylcarbamoyl]-propyl}-pyrrolidin-1-yl)-2-methoxy-4-oxo-butyl]-methyl-carbamoyl}-2-methyl-propyl)-2-dimethylamino-3-methyl-butyramide
of the present invention were examined by i.v. administration in mice. The respective MTD values of the compounds were 14, 18, 10, 8, 8, 2 and 2 mg/kg.
Thus the compounds of the invention are therapeutically active, producing regression or remission of solid tumors.
The present invention concerns also the use of a compound of formula (I) for the preparation of pharmaceutical compositions, preferably for the preparation of pharmaceutical compositions for the treatment of cell proliferative disorders, more preferably for the preparation of pharmaceutical compositions for the treatment of cancer, and most preferably for the treatment of colorectal cancer, lung cancer, breast cancer, stomach cancer, cervical cancer and bladder cancer.
Another aspect of the present invention is a method for treating a cell proliferative disorder comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I).
In accordance with the present invention, treatment of cancers is accomplished by administering a compound of the invention systemically to a patient in an amount effective to treat the cancer. By inhibiting growth of cancer (carcinoma) cells is meant stopping growth, causing apoptosis, or causing differentiation, or otherwise changing the nature of the cell to render it innocuous. The compound may also be administered prophylactically, for example to a person at risk for cancer, or a person who has already undergone effective treatment generally in a lower dosage than for treatment. The amount of compound used is dependent on the type of cancer, the amount and size of the tumors and on the requirements of the patient. In general a daily dosage of about 0.1 mg/kg to about 100 mg/kg of body weight, preferably about 20 mg/kg to about 80 mg/kg is a helpful basic range, which may be varied by the skilled practitioner depending on the characteristics and requirements of the patient and his condition. The treatment is typically carried out for a period of about three months, but this depends on the patient""s condition and the practitioner""s judgement. In prophylactic administration, the duration of administration again depends on the patients condition and the practitioner""s plan, but will generally continue for a longer period of time than three months. For the treatments given above, the compound of the invention is administered systemically as a composition containing the compound of the invention, and a pharmaceutically acceptable carrier compatible with said compounds. In preparing such composition, any conventional pharmaceutically acceptable carrier can be used. Generally the preferred unit dosage form is tablets or capsules, which can be administered once or twice daily depending upon the weight and size of the patient. The compounds of this invention may be administered as the sole treatment, or may be used in conjunction with other chemical or biochemical treatments or with radiation or surgery.
The pharmaceutical compositions of this invention can be made up in any conventional form including: (a) a solid form for oral or suppository administration such as tablets, capsules, pills, powders, granules, and the like; (b) sterile, typically aqueous solution or suspension form for intravenous or parenteral administration and (c) preparations for topical administration such as solutions, suspensions, ointments, creams, gels, micronized powders, aerosols and the like. The pharmaceutical compositions may be sterilized and/or may contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, salts for varying the osmotic pressure, and/or buffers.
The compounds of the invention are especially useful in pharmaceutically acceptable oral modes. These pharmaceutical compositions contain one or more compounds of the invention or its pharmaceutically acceptable salts and its pharmaceutically acceptable hydrolyzable esters in association with a compatible pharmaceutically acceptable carrier material. Any conventional carrier material can be used. The carrier material can be an organic or inorganic inert carrier material suitable for oral administration. Suitable carriers include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene-glycols, petroleum jelly and the like. Furthermore, the pharmaceutical preparations may contain other pharmaceutically active agents. Additional additives such as flavoring agents, preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding.
The pharmaceutical preparations can be made up in any conventional oral dosage form including a solid form for oral administration such as tablets, capsules, pills, powders, granules, and the like. A preferred oral dosage form comprises tablets, capsules of hard or soft gelatin, methylcellulose or of another suitable material easily dissolved in the digestive tract. The oral dosages contemplated in accordance with the present invention will vary in accordance with the needs of the individual patient as determined by the prescribing physician.
General Synthesis Schemes
The compounds of the present invention may be prepared by a skilled person by condensing an acid of the formula (II), 
wherein R1 and R2 are as defined above. Preferably R1 and R2 are each independently alkyl, more preferably (C1-C6)-alkyl, and most preferably (C1-C4)-alkyl;
with a compound of the formula (III), 
xe2x80x83wherein R3, R4, R5 and n are as defined above.
Compounds of formula (I) may be prepared by condensing an acid of the formula (II) with a compound of formula (III) in the presence of a condensing agent, followed, if necessary, by removal of protecting group(s) and/or a salt formation, if necessary.
Alternatively, compounds of formula (I) can be prepared by
condensing an acid of the formula (IV), 
xe2x80x83wherein R1 is hydrogen or alkyl, preferably (C1-C6)-alkyl, and most preferably (C1-C4)-alkyl; and R6 is a protecting group selected from t-butoxycarbonyl, carbobenzyloxy or 9-fluorenylmethoxycarbonyl (Fmoc),
with a compound of the formula (III), 
xe2x80x83wherein R3, R4, R5 and n are the same as defined above, in the presence of a condensing agent if necessary, by removal of protecting group(s) and/or a salt formation, if necessary. The condensing agent may be e.g. dicyclohexylcarbodiimide (DCC), diphenyl phosphorylazide (DPPA), diethyl phosphorocyanide (DEPC), benzotriazol-1-yloxy-tris(dimethylamino) phosphonium hexafluorophosphate (BOP reagent), or the like in an inert solvent such as, for example, halogenated aliphatic hydrocarbon such as chloroform and dichloromethane, ethylacetate, tetrahydrofuran (THF), dimethylformamide (DMF) or acetonitrile, if necessary in the presence of an organic base such as, for example, triethylamine or diisopropylethylamine (DIPEA).
The compound of the present invention represented by the formula (I) wherein either R1 or R2 is a hydrogen atom can be prepared by condensing a tripeptide fragment of the following formula (IV) 
wherein R1 is hydrogen or alkyl, preferably (C1-C6)-alkyl, and most preferably (C1-C4)-alkyl; R6 is a protecting group, e.g. selected from t-butoxycarbonyl (Boc), carbobenzyloxy (Z) or 9-fluorenylmethoxycarbonyl (Fmoc) group;
with a fragment of the following formula (III) 
xe2x80x83wherein R3, R4, R5 and n are as defined above by using a condensing agent.
The condensing agent may be, e.g., dicyclohexylcarbodiimide (DCC), diphenyl phosphorylazide (DPPA), diethyl phosphorocyanide (DEPC), BOP reagent, or the like in an inert solvent such as, for example, halogenated aliphatic hydrocarbon such as chloroform and dichloromethane, ethylacetate, tetrahydrofuran (THF), dimethyl-formamide (DMF) or acetonitrile, if necessary in the presence of an organic base such as, for example, triethylamine or diisopropylethylamine (DIPEA) at a temperature between xe2x88x9210xc2x0 to 50xc2x0 C., preferably 0xc2x0 C. to room temperature, and then the coupling product is deprotected by the procedures known to those in the art, e.g. by basic or acidic hydrolysis, hydrogenolysis or treatment with fluoride anion.
Compounds of formula (III) are novel and are also an object of this invention.
Another embodiment of the present invention concerns the preparation of compounds of formula (III).
Compounds of formula (III) 
wherein R3, R4, R5 and n are as defined above, can be prepared according to the following synthetic scheme 1. 
According to scheme 1, compounds of formula (III) are prepared from compound of formula (V), wherein R3 is hydrogen or alkyl, preferably (C1-C6)-alkyl, and most preferably (C1-C4)-alkyl; R7 is a protecting group selected from t-butoxycarbonyl, carbobenzyloxy or Fmoc group, prepared from N-Boc-prolinal by known methods (Heterocycles, 36 (9) 2073-2080, 1993), by reacting with a compound of formula (VI), a commercially available compound as the salt or prepared from the corresponding mercaptane with a base such as sodium hydroxide, sodium hydride, sodium carbonate, sodium hydrogen carbonate, potassium hydroxide, potassium hydride or potassium t-butoxide, lithium hydroxide, lithium hydride, methyl lithium or n-butyl lithium, by conventional methods, conveniently in an inert organic solvent, such as tetrahydrofuran, acetonitrile, methanol, ethanol or DMF, at a temperature from about xe2x88x9240xc2x0 C. to the reflux temperature of the solvent to form a corresponding intermediate of formula (VII) wherein R3 and R4 are as defined in the present invention; R7 is a protecting group selected from t-butoxycarbonyl, carbobenzyloxy or Fmoc group. Potassium thiomethoxide, in particular, can be alternatively prepared conveniently from the reaction of methyl thioacetate with potassium ethoxide in situ instead of using methylmercaptane gas.
The Michael addition of potassium thioalkoxide in the presence of a proton source such as an alcohol or phenol, preferably phenol, proceeds smoothly at room temperature, giving the desired stereoisomers regarding sulfur group and R3 in good yield and stereoselectivity. For example, the reaction of the compound formula (V), where R3 is methyl, R7 is t-butoxycarbonyl group and the configuration of the proline 2-position is S, with potassium thiomethoxide or thioethoxide in the presence of phenol gives predominantly (2S)-2-[(1R,2S)-2-ethoxycarbonyl-1-methyl or ethyl-sulfanyl-propyl]-pyrrolidine-1-carboxylic acid tert-butyl ester.
The intermediate of formula (VII), wherein R3, R4 and R7 are as defined above, is hydrolyzed, if necessary, by conventional methods and then reacted with an alcohol or an amine, conveniently using an aforementioned condensing agent in an inert organic solvent, such as a halogenated aliphatic hydrocarbon, tetrahydrofuran, acetonitrile, or DMF, at a temperature of from about xe2x88x9220xc2x0 C. to the reflux temperature of the solvent, preferably from 0xc2x0 C. to room temperature, to form a corresponding compound of formula (IX) wherein R3, R4 and R5 are as defined in the present invention; R7 is a protecting group selected from t-butoxycarbonyl, carbobenzyloxy or Fmoc group and n is an integer of 0.
The compound of formula (IX), wherein R3, R4 and R5 are as defined above and n is 0; R7 is a protecting group selected from t-butoxycarbonyl, carbobenzyloxy or Fmoc group, can be optionally oxidized with m-chloroperbenzoic acid (mCPBA) by conventional methods, conveniently in an inert organic solvent, such as a halogenated aliphatic hydrocarbon, at a temperature of from about xe2x88x9240xc2x0 C. to the reflux temperature of the solvent to form a corresponding sulfoxide or sulfone derivative of formula (IX), wherein R3, R4 and R5 are as defined in the present invention and n is an integer of 1 or 2; R7 is a protecting group selected from t-butoxycarbonyl, carbobenzyloxy or Fmoc group.
The compound of formula (I), wherein R1, R2, R3, R4 and R5 are as defined in above and n is 0, can be also optionally oxidized with mCPBA by conventional methods, conveniently in an inert organic solvent, such as a halogenated aliphatic hydrocarbon, at a temperature of from about xe2x88x9240xc2x0 C. to the reflux temperature of the solvent to form a corresponding sulfoxide or sulfone derivative of formula (I) wherein R1, R2, R3, R4 and R5 are as defined above ad n is 1 or 2.
Alternatively, the compound of formula (I), wherein R1, R2, R3, R4 and R5 are as defined above, provided that either R1 or R2 is hydrogen, and n is 1 or 2, can be also prepared by oxidation of the coupling product obtained from (IV), wherein R1 is alkyl group; R6 is a protecting group selected from t-butoxycarbonyl, carbobenzyloxy or Fmoc group, and (III), wherein R3, R4 and R5 are as defined above and n is 0, with mCPBA followed by deprotection known to those in the art, e.g. by basic or acidic hydrolysis, hydrogenolysis or treatment with fluoride.
The compound of formula (IX), wherein R3, R4, R5, n are as defined above and R7 is a protecting group selected from t-butoxycarbonyl, carbobenzyloxy or Fmoc group, is deprotected with trifluoroacetic acid (TFA) in an inert solvent such as a halogenated aliphatic hydrocarbon or without solvent at a temperature of from about xe2x88x9220xc2x0 C. to the reflux temperature of the solvent, preferably from 0xc2x0 C. to room temperature, to form a corresponding compound of formula (III) as the TFA salt.